Fungal Diseases

SOME EXISTENTIAL QUESTIONS

• WHY ARE WE HERE?

• WHY ARE WE SO HOT?

• WHY DO WE EAT SO MUCH?

• WHY ARE MAMMALS THE DOMINANT LARGE ANIMALS?

Miasmic Theory of Disease

19th Century Representation of Cholera Spreading as Foul Air

Girolamo Fracastoro by Titian

Germ Theory of Disease (1850-1880)

GERM THEORY OF DISEASE WAS A REVOLUTION IN THOUGHT

• CAUSAL ASSOCIATION BETWEEN CERTAIN MICROBES AND DISEASE

• PROVIDED ACTIONABLE INFORMATION TO REDUCE MORTALITY FROM INFECTIOUS DISEASES

JAMA 1999:281(1):61-66

SANITATION

SERUM THERAPY

VACCINES

Forgotten facts: causality between germs and disease was established first with fungi

1899 FIN DE SIECLE: FUNGAL DISEASES EXTREMELY RARE

TUBERCULOSIS

DIPHTHERIA

POLIO

ANTHRAX, PLAGUE

VARIOLA

MEASLES MALARIA

TRYPANOSOMESHELMINTHS

TYPHUS

SYPHILIS

1999 FIN DE SIECLE: FUNGAL DISEASES DISTRESSINGLY COMMON

Candida albicans Cryptococcus neoformans

Coccidioides immitis Aspergillus spp.

WHAT HAPPENED? HOSTS CHANGED…MICROBE-FOCUSED VIEWS OF

MICROBIAL PATHOGENESIS COULD NOT COPE WITH THE CHANGE

THE HOST WAS ALTERED!

MEDICAL PROGRESS

A CATACLYSM

ANTIBIOTICS IMMUNOSUPPRESANTS CHEMOTHERAPY ICU’s

Park et al. AIDS 2009

106 Cases/Y

IV Lines

Fungal Diseases are:Not ReportableUnderestimatedPoorly studiedNeglectedNot PrioritiesWHY????

The late 20th Century Crisis

1900: Two types of microbes: pathogens & non-pathogens (fungal diseases rare)

1950: ‘Non-pathogens’ begin to be associated with disease in setting of steroid use, antimicrobial use and early chemotherapy (fungal diseases emerge)

1970: Concept of ‘opportunism’ emerges to explain ‘non-pathogen’ causing disease (fungal diseases seen as opportunistic)

1990: ‘non-pathogens’ have become pathogens (fungal diseases common).

Microbe-centric theories of pathogenesis cannot explain changes

Crisis leads to a messy lexicon - examples

Candida albicans ‘commensal’ in asymptomatic humans

‘primary pathogen’ in vaginal candidiasis

‘opportunistic pathogen’ in AIDS

Pneumococcus ‘colonizer’ in asymptomatic humans

‘primary pathogen’ in elderly pneumonia

‘opportunistic pathogen’ in AIDS pneumonia

Aspergillus spp. ‘saprophyte’ in asymptomatic humans

‘primary pathogen’ in allergic aspergillosis

‘opportunistic pathogen’ in transplant patients

1995-1999

Liise-anne Pirofski and I are asked to create a new graduate course at the Albert Einstein College of Medicine on Microbial Pathogenesis

Designing a course on an overarching concept of microbial virulence was impossible since none was available

Began to experiment with various concepts, which led to many discussions.

By 1999 class notes had converged into a new framework and we realized that we had something new.

1999 – First Publication

Thought and Theoryare not highly valuedin current biologicalsciences. Only way to get this published was yo hide it inside a review

Lexicon of microbial pathogenesis was a mess

The ‘Damage-Response Framework’ of Microbial Pathogenesis

Disease is (only) one outcome of an interaction between a microbeand a susceptible host.

The relevant measure of host-microbe interaction is host damage due to host or microbial factors, or both.

Microbes are defined by their state in a host, not by their traits or what they ‘do’.

Casadevall & Pirofski, Nature Micro Rev 2003

Fungal Diseases occur at both horns of the parabola

CryptococcosisDisseminated aspergillosisDisseminated Candidiasis

Fungal asthmaAllergic pulmonary aspergillosisVaginal candidiasis

‘Damage-Response Framework’:Five Consequences

1. SHIFTS DEBATE FROM ‘PATHOGENS’ vs ‘NON-PATHOGENS’ TO OUTCOME OF THE INTERACTION

2. THERE ARE NO ‘PATHOGENS’, ‘COMMENSALS’, ‘OPPPORTUNITSTS’, ‘SAPROPHYTES’: THERE ARE ONLY MICROBES AND HOSTS

3. VIRULENCE IS ONE OUTCOME OF THE INTERACTION OF MICROBE AND HOSTS (OTHER OUTCOMES ARE COMMENSALISM, SYMBIOSIS, INDIFERENCE, ETC)

4. OUTCOMES OR STATES ARE CONTINOUS ARE DIFFER ONLY IN THE AMOUNT OF DAMAGE INCURRED BY THE HOST

5. SEES VIRULENCE AS AN EMERGENT PROPERTY

A HOST IS AN ENTITY THAT HOUSES AN ASSOCIATED MICROBITA/MICROBIOMEAND INTERACTS WITH MICROBES SUCH THAT THE OUTCOME RESULTS IN DAMAGE,BENEFIT OR INDIFFERENCE THUS RESULTING IN THE STATES OF SYMBIOSIS,COLONIZATION, COMMENSALISM, LATENCY OR DISEASE

WHAT IS A HOST?

NOT A HOST

NATURE 2014Nature 2014

Could a mathematical formalism be developed that would account for both host + microbe that

also illustrated the continuity of interactions?

PATHOGENIC POTENTIAL OF A MICROBE

• NEW CONCEPT: THE ‘PATHOGENIC POTENTIAL’

• DEVELOPED A FORMALISM TO QUANTIFY IT

• PP=(Fs/I)(𝟏𝟎𝑴)

• Fs IS THE FRACTION SYMPTOMATIC, I IS THE INOCULUM AND M IS MORTALITY FRACTION

Microbe Mortality Innoculum PP Reference

Theoretical Max. 1.0 1 1.0 x 101 This work

Franciscella tularensis 0.5 1-2 2.5 x 100 (13)

Bacillus anthracis 0.5 2.6 1.2 x 100 (14)

Brucella suis 0.5 3.8 8.3 x 10-1 (15)

Toxoplasma gondii 0.5 15 2.1 x 10-1 (16)

Coccidioides immitis 0.5 16.7 1.9 x 10-1 (17)

Klebsiella pneumoniae 0.5 17.9 1.8 x 10-1 (18)

Streptococcus pneumoniae 0.5 30 1.1 x 10-1 (19)

Yersinia pestis 0.5 37 8.5 x 10-2 (20)

Cryptococcus neoformans 0.5 51 6.2 x 10-2 (21)

Vibrio vulnificus 0.5 75 4.2 x 10-2 (22)

Herpes Simplex Virus 0.5 219 1.4 x 10-2 (23)

Escherichia coli 0.5 1 x 103 3.2 x 10-3 (24)

Candida albicans 0.5 6.6 x 103 4.5 x 10-4 (25)

Murine Cytomegalovirus 0.5 5.0 x 104 6.3 x 10-5 (23)

Aspergillus fumigatus 0.25 6.0 x 104 3.0 x 10-5 (26)

Group B Streptococcus 0.5 6.3 x 104 5.0 x 10-5 (27)

Murine adenovirus 0.5 1.0 x 105 3.2 x 10-5 (28)

Listeria monocytogenes 0.5 2.4 x 105 1.3 x 10-5 (29)

Nocardia asteroides 0.5 8.5 x 105 3.7 x 10-6 (30)

Shigella sonnei 0.5 1.6 x 106 2.0 x 10-6 (31)

Naegleria fowleri 0.75 5.0 x 106 1.1 x 10-6 (32)

Bacillus cereus 0.5 1.0 x 107 3.1 x 10-7 (14)

Staphylococcus saprophyticus 0.5 2.7 x 107 1.2 x 10-7 (33)

Bacillus thuringiensis 0.5 1.1 x 107 2.9 x 10-7 (14)

Pseudomonas aeruginosa 0.5 5.0 x 107 6.3 x 10-8 (34)

Legionella pneumophila 0.5 6.7 × 107 4.7 x 10-8 (35)

Staphylococcus epidermitis 0.5 6.0 x 107 5.3 x 10-8 (33)

Staphylococcus aureus 0.5 1.0 x 108 3.1 x 10-8 (36)

Hemophilus influenza b 0.5 2.0 x 108 1.6 x 10-8 (37)

+ mucin2 0.5 3.4 x 104 9.3 x 10-5 (37)

Enterococcus faecalis 0.5 2.6 x 108 1.2 x 10-8 (38)

CASADEVALL A (2017) mSphere

PATHOGENIC POTENTIAL IS A FUNCTION OF THE HOST

Microbe Mouse Strain Mortality IInoculum PP

L. monocytogenes C57B1/6 0.5 9.0 x 105 3.5 x 10-6

B10.D2 0.5 2.2 x 105 1.4 x 10-5

B10.A 0.5 2.2 x 105 1.4 x 10-5

BALB/c 0.5 3.9 x 103 8.8 x 10-3

CBA 0.5 5.0 x 103 6.3 x 10-4

A/WySn 0.5 8.0 x 103 4.0 x 10-4

(C57Bl/6 x BALB/c)F1 0.5 3.4 x 104 9.3 x 10-5

B. anthracis A/J 0.5 2.6 x 100 1.2 x 100

C3H/HeJ 0.5 5.6 x 100 5.6 x 10-1

BALB/cJ 0.5 6.6 x 100 4.8 x 10-1

C58J 0.5 9.0 x 100 3.5 x 10-1

C57BL/6J 0.5 1.4 x 101 2.3 x 10-1

C57L/J 0.5 2.2 x 101 1.4 x 10-1

Sendai virus 129/ReJ 1 0.5 3.2 x 100 1.0 x 10-1

SWR/J 0.5 5.0 x 102 6.3 x 10-3

C58/J 0.5 1.5 x 103 2.1 x 10-3

C57BL/6J 0.5 2.5 x 104 1.3 x 10-4

SJL/J 0.5 1.0 x 105 3.2 x 10-5

C. immitis BALB/cAnN 0.5 4.6 x 101 6.9 x 10-2

C57BL/10N 0.5 5.9 x 102 5.4 x 10-3

C57BL/6N 0.5 6.8 x 102 4.7 x 10-3

DBA/2NX1 0.5 1.8 x 105 1.8 x 10-5

Casadevall (2017) mSphere

Concept of ‘pathogenic potential’ beginning to be used instead of ‘virulence’

The Fungal Kingdom

• > 6 million species

• Includes major pathogens of plants, insects, invertebrates and ectothermic vertebrates

• Fungi currently devastating major ecosystemsBats devastated by ‘white nose syndrome’

Catastrophic amphibian declines from Batrachochytrium dendrobatidis

Salamanders declines in Europe from Batrachochytrium salamandrivorans

Snakes in North America

• Mammals are remarkably resistant!

Few fungal species are pathogenic for humans

Host Associated

Aspergillus spp.

Dermatophytes Histoplasma spp.Candida spp.

Cryptococcus spp.

Blastomyces spp.

Coccidioides spp.

Environment

Pneumocystis spp.

Fungi are devastating entire ecosystems

Corals Frogs Rice

Salamanders Turtles Wheat

Puccinia graminis

Gorgonia ventalina B. dendrobatidis

B. salmandrivorans Fusarium solani

Magnaporte oryzae

Snakes

O. ophiodiicola

Rabbit Experiments Suggest that Remarkable Resistance of Mammals is a Result of Adaptive Immunity PLIUS High Temperature (J. Perfect and others)

+SYSTEMICINFECTION

TESTICULARINFECTION

T = 40-41 C

HEALTHYRABBITTHAT CLEARSINFECTION

CHRONICORCHITIS

C. neoformans

SYSTEMICINFECTION +STEROIDS

EMERGING PATTERN:

ADAPTIVE IMMUNITY + ENDOTHERMY = RESISTANCE TO FUNGAL DISEASES

White Nose Syndrome in Bats

What makes mammals so special?

• Adaptive Immunity

• Endothermy

• Being ‘hot’ is a tradeoff

• Birds also resistant to fungal diseases

• Primitive mammals (e.g. platypus)

susceptible

Robert & Casadevall, JID 2009

Vicent Robert

 

B(T) µ M3

4e-Ei

kT

 

F(T) µ F0[1- (1- s)T ]

Cost

Benefit

Maxima: 36.7 C Bergman & Casadevall, mBio 2010

Aviv Bergman

The cost of the mammalian lifestyle

• Mammalian lifestyle is very expensive

• Mammals very minor class until 65 mY ago

• Great mammalian radiation after K/T event

• How did this unfavorable lifestyle become dominant?

Could the explanation be in the distant past? When mammals emerged?

1,000,000 500,000 PRESENT

Ascomycetes (H. capsulatum,

C. immitis, B. dermatides, Candida, etc)

Basidiomycetes (C. neoformans)

LANDANIMALS

K-T EVENT

AMPHIBIANS

REPTILES

BIRDS

MAMMALS ENDOTHERMS

ECTOTHERMS

DINOSAURSx

PROTOZOA

AGE OF MAMMALS BEGINS AFTER THE K-T EVENT

FUNGI

CRETACEOUS TERTIARY

REPTILIAN MEGAFAUNA MAMMALIAN MEGAFAUNA

SMALL MAMMALS THAT ATE THE EGGS?

WHAT KILLED THE DINOSAURS?(it depends who you ask)

MASSIVE VOLCANISM FROM DECCAN TRAPS 65 MILLION YEARS AGOA ROCK FROM OUTER SPACE

Current Biology 2012

THE CRETACEOUS WORLD AND ITS END

REPTILIAN MEGAFAUNA YUCATAN IMPACT

A WARM, VOLCANIC AND FORESTED PLANET THE ATLANTIC WAS TINY…

THE POST-IMPACT WORLD

• FIRES, SMOKE, DUST OBSCURES SUN• PHOTOSYNTHESIS SHUT DOWN FOR > 6 MONTHS• GLOBAL TEMPERATURES DROP

KT EVENT CATASTROPE = GLOBAL FUNGAL COMPOST

VEGETATION DIEOFF = FUNGAL PROLIFERATIONFUNGAL PROLIFERATION = SPORE PROLIFERATIONSPORE PROLIFERATION = LARGE INOCULAENDOTHERMY = RESISTANCE TO FUNGAL DISEASE

FUNGAL GEN BIOL 2005

THE POST IMPACT WORLD

REPTILES FIGHT OFF FUNGALINFECTIONS WITH INDUCED

FEVERS BUT THERE WAS NO SUN

CHINESE EARTH SCIENCES 2008

REPTILIAN EGGS SUSCEPTIBLE TO FUNGAL DISEASES

Courtesy of NIAID

HYPOTHESIS: A FUNGAL FILTER SELECTED FOR MAMMALS OVER REPTILES

AGE OF REPTILES

AGE OF MAMMALS

GLOBAL COMPOST

ENDOTHERM SELECTION

AGE OF MAMMALS

SCIENCE 2003

FUNGALSELECTIONCREATES A

FILTER FOR THEEMERGENCE OF MAMMALS

REPTILIAN MEGAFAUNA MAMMALIAN MEGAFAUNA

Courtesy of NIAID

How do we know that there were fungal pathogens in the distant past?The Cryptococcal Intracellular Pathogenic Strategy dates to Cretaceous

H99 (VN1)WM179 (VG1)R265 (VG2a)WM161 (VG3)

Intracellular replicationIntracellular capsular enlargementPhagosomal acidificationPhagosomal leakageNon-lytic exocytosisMacrophage transcriptional profile

C. neoformans Strains

Diverged50-100 mYAgo

A Cryptococcus Pangean Ancestor had pathogenic potential

Cryptococcus spp. complexesSeparated 80-100 myaBreakup of Gondwana 100 myaAll have same virulence factorsPangean ancestor must have been similarConservation of traits by similar pressures?

Requirements for Fungal Human Pathogenicity

Thermotolerance

• Host associated such as Candida spp. already thermotolerant

• Only 6% of species in environment can tolerate > 37 oC (Robert & Casadevall JID 2009)

• Only a few ‘major’ pathogenic fungi (Aspergillus, Cryptococcus, Histoplama, Sporothrix, Coccidioidesspp.)

Survival in host and replication

• “Virulence factors”

• Survive, replicate and evade immune mechanisms

• Highly varied…

Capsules

Toxins

Antioxidant systems

Intracellular replication

Stress resistance

etc., etc., etc.

Cryptococcus neoformans

POLYSACCHARIDE CAPSULE

MELANIN IN CELL WALL

For C. neoformans capsule and melanin make largest contribution to virulence composite

~26%

~14%

CONUNDRUM: C. NEOFORMANS IS A NON-SPECIFIC PATHOGEN WITH A VERY SOPHISTICATED CELLULAR PATHOGENIC STRATEGY..WHAT IS GOING ON HERE?

PLANTS

SOPHISTICATION MANY HOSTS - NONSPECIFICITY

WHY DOES A SOIL ORGANISM WITHNO NEED FOR AN ANIMAL HOST HAVEA SOPHISTICATED VIRULENCE STRATEGY?

HOW DOES A SOIL ORGANISM WITH NONEED FOR ANY HOST MANAGES TO SUBVERTSUCH DIVERSE HOST DEFENSES?

Why do soil organisms have mammalian virulence factors?

• C. neoformans virulence factors capsule, melanin and phospholipase allow survival in confrontation with amoeba

• Similar findings with other pathogenic fungi

• ‘Ameboid predator hypothesis’Capacity for virulence is accidental (Casadevall & Pirofski 2007)

Front. Cell. Infect. Microbiol 2017

Macrophages sustain damage across many critical cellular systems

Carolina Coelho

CRISPR-CAS9 SCREEN FOR RESISTANCE GENESNETWORK ANALYSIS OF MACROPHAGE GENESSHOW MITOCHONDRIAL AND RIBOSOMAL GENES ARE DISPROPORTIONALY AFFECTED BYC. NEOFORMANS INFECTION

MITOCHONDRIA

RIBOSOMAL

UNPUBLISHED

New Roles for Old Cryptococcal Virulence Factors

Virulence factor Known Role New Role

Urease Brain invasion Intracellular pathogenesis

Nutrient acquisition

Polysaccharide Capsule Antiphagocytic Phagosome pH Regulation

Antioxidant Flotation Device

Melanin Antioxidant Heat capture

→

C. neoformans Urease

Urease role is as a nutritional enzyme

Plos Pathogens 2018

An example of a nutritional enzyme affecting virulence

Mechanism of urease action intracellularly

Urease changes phagosomal pH …which affects phagosomal leakage …which affects mode of death

Plos Pathogens 2018

A New Role for Melanin

Current Biology 2018

Laccase catalyzes synthesis of pigments with different colors in C. neoformans

Current Biology 2018

Pigmented C. neoformans harvest energy from light in cold room to grow faster

Current Biology 2018

Microbial Pigments in Energy Harvest and Global Warning

Pigmented microbes colonizeMeltwater and enhance melting

Black Mountains of Antarctica where pigment comes from microbes

GEOGRAPHIC FACTS: MOST ENDEMIC MYCOSES OCCUR IN TROPICAL AND SUB-TROPICAL REGIONS

AVERAGE FUNGAL THERMAL TOLERANCES OVER PAST 30 YEARS

Robert et al BMC Biology (2015)

Summary

Fungal diseases cause tremendous suffering for humanity

Fungal disease are neglected, understudied, underfunded…

Most humans do not fear fungal diseases

Remarkable mammalian resistance to fungi due to endothermy + acquired immunity

Late 20th century emergence of fungi as major pathogens reflect changes that undermined immunity

Global warming is next big threat as it will reduce protective thermal gradient and new fungal diseases are likely to occur

Some Answers to Existential Questions from a FungalPoint of View

• WHY ARE WE HERE?KT EVENT AND FUNGAL SELECTION

• WHY ARE WE SO HOT?TO KEEP THE FUNGI AWAY

• WHY DO WE EAT SO MUCH?TO MAINTAIN HIGH TEMPERATURE

• WHY ARE MAMMALS THE DOMINANT ANIMALS?FUNGAL SELECTION KEPT DOWN THE REPTILES

NO SECOND AGE OF REPTILES!